Development of monoclonal antibody based sandwich ELISA for the rapid detection of pathogenic Vibrio parahaemolyticus in seafood

International Journal of Food Microbiology 145 (2011) 244–249

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Development of monoclonal antibody based sandwich ELISA for the rapid detection of pathogenic Vibrio parahaemolyticus in seafood Ballamoole Krishna Kumar 1, Pendru Raghunath 1, Devananda Devegowda, Vijay Kumar Deekshit, Moleyur Nagarajappa Venugopal, Iddya Karunasagar, Indrani Karunasagar ⁎ Department of Fishery Microbiology, Karnataka Veterinary, Animal and Fisheries Sciences University, College of Fisheries, Mangalore 575 002, India

a r t i c l e

i n f o

Article history: Received 2 June 2010 Received in revised form 23 November 2010 Accepted 28 December 2010 Keywords: Vibrio parahaemolyticus Thermostable direct hemolysin (TDH) TDH-related hemolysin (TRH) Monoclonal antibody (mAb) Enzyme-linked immunosorbent assay (ELISA)

a b s t r a c t Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are considered important virulence factors of Vibrio parahaemolyticus and strains producing either of these or both are considered pathogenic. In this study, we generated monoclonal antibodies (mAbs) against purified TRH recombinant protein of pathogenic V. parahaemolyticus. Sandwich enzyme-linked immunosorbent assays (ELISA) using the hybridoma clone 4B10 showed higher sensitivity of detection compared to other clones. Using mAb 4B10 based sandwich ELISA, we could detect pathogenic V. parahaemolyticus in 41.18% (14 out of 34) of the seafood samples analyzed. PCR targeting the toxR gene showed the presence of V. parahaemolyticus in 64.7% (22 out of 34) seafood samples. Further, PCR targeting the virulence genes showed that 6 seafood samples harboured the tdh gene while 9 harboured the trh gene indicating the presence of pathogenic V. parahaemolyticus. Our results show that mAb 4B10 sandwich ELISA developed in this study could be used as a rapid method for screening seafood samples for the presence of pathogenic V. parahaemolyticus. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Vibrio parahaemolyticus is a Gram negative, halophilic bacterium, autochthonous to water and sediments of marine and estuarine ecosystem. The incidence of gastroenteritis due to V. parahaemolyticus was first reported in Japan during the 1950s (Fujino et al., 1951, 1953) and since then, cases have been reported from several parts of the world. The pathogenesis of this organism has been associated with thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) coded by the tdh and trh genes respectively (Honda et al., 1987a, 1987b; Honda and Iida, 1993). Therefore, the strains bearing tdh and/or trh genes are considered as pathogenic (Nishibuchi and Kaper, 1995). The establishment of an effective control programme for vibrios in seafood necessitates reliable, accurate and sensitive methods to assess the presence of pathogenic vibrios in seafood. Conventional culture based techniques are slow, laborious and often require several days. Moreover, these assays, may fail to detect strains of bacteria which are present in the samples at very low levels (Aono et al., 1997). β-hemolysis on high salt blood agar, Wagatsuma agar is used to detect V. parahaemolyticus producing TDH, but there is no phenotypic test for the detection of TRH. PCR has been employed for the detection of V. parahaemolyticus from both clinical and environmental samples. These methods target the genes ⁎ Corresponding author. Tel./fax: + 91 824 2246384. E-mail addresses: [email protected], [email protected] (I. Karunasagar). 1 These authors contributed equally to this work. 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.12.030

encoding virulence determinants and also species specific markers that include tdh, trh, tlh and toxR (Taniguchi et al., 1985; Tada et al., 1992; Karunasagar et al., 1996; Bej et al., 1999; Kim et al., 1999; Dileep et al., 2003). Though the PCR is more sensitive than conventional culture methods, inability of PCR to discriminate between live and dead bacteria due to the persistence of DNA after cell death is considered as a major drawback (Hayashi et al., 2006). Hence specific detection of pathogenic V. parahaemolyticus is of utmost importance from the public health and seafood industry point of view. Monoclonal antibody based tests are considered as powerful tools for the identification of microorganisms from various sources. The first report on the production of monoclonal antibody against pathogenic V. parahaemolyticus (TDH and TRH) was made by Honda et al. (1989, 1990) wherein they found that the developed enzymelinked immunosorbent assay (ELISA) was highly sensitive for the identification of TDH and TRH of pathogenic V. parahaemolyticus from clinical samples. In this study, we report, the production of monoclonal antibodies (mAbs) against TRH of V. parahaemolyticus and their application in ELISA to detect pathogenic V. parahaemolyticus (tdh and/or trh) from seafood enrichment cultures. 2. Materials and methods 2.1. Bacterial strains The bacterial strains used in this study are listed in Table 1. All isolates used in this study were revived from stock culture preserved

B.K. Kumar et al. / International Journal of Food Microbiology 145 (2011) 244–249

2.4. Characterization of monoclonal antibodies

Table 1 List of bacterial strains used in this study. Name of organism

Recombinant TRH Vibrio parahaemolyticus (trh+) (n = 21) V. parahaemolyticus (tdh+) (n = 03) V. parahaemolyticus (tdh+and trh+) (n=06) V. parahaemolyticus (non-pathogenic) (n = 05) V. cholerae (n = 05) V. harveyi (n = 03) V. mimicus (n = 02) V. alginolytiucs (n = 03) V. anguillarum (n = 02) V. damsella (n = 02) Aeromonas hydrophila (n = 02) A. veronii biovar veronii (n = 03) Escherichia coli (n = 05) Salmonella (n = 06)

245

1B10

4B10

4C12

0.814 ± 0.233 0.440 ± 0.184

1.137 ± 0.054 0.718 ± 0.348

0.701 ± 0.100 0.503 ± 0.162

The generated mAbs were tested for its specificity using all bacterial strains mentioned in Table 1 by plate ELISA. Western blotting was performed by the procedure of Towbin et al. (1979) using supernatants from positive clones for the reactivity with recombinant TRH protein.

0.355 ± 0.053

0.684 ± 0.391

0.310 ± 0.007

2.5. Production of monoclonal antibodies

0.444 ± 0.163

0.665 ± 0.227

0.412 ± 0.252

−

−

−

− − − − − − − − − −

− − − − − − − − − −

− − − − − − − − − −

Absorbance values of mAb based sandwich ELISA (mean OD ± SD)

Note: Positive–negative threshold (negative index) was calculated using the formula, negative OD × 2. More than 2 times that of a negative control considered as positive. The OD of non-pathogenic V. parahaemolyticus and other bacterial strains was below the threshold value 0.280 (ranging from 0.120 to 0.264). “−” means negative for pathogenic V. parahaemolyticus (below the threshold value of 0.280).

at − 80 °C (Sanyo, Japan) in 30% glycerol broth at Department of Fishery Microbiology, College of Fisheries, Mangalore. These strains were grown in 2 mL of Luria–Bertani broth (HiMedia, India) at 37 °C overnight with shaking. 2.2. Cloning and expression of the trh gene The trh gene was amplified from the V. parahaemolyticus using primers (5′–3′) trh5-TTGCTATTGGTTTCAATATT and trh6-AATTTGTGACATACATTCAT. The amplified PCR products were purified using QIAquick PCR purification kit (Qiagen, USA). Purified products were analyzed by 1.5% agarose gel electrophoresis. Purified PCR product was ligated to pQE30 UA vector (Qiagen, USA) and transformed into SG13009 E. coli cells. Recombinant clones were confirmed by PCR using gene specific primers. TRH expression in recombinant clones was done by inducing with a final concentration of 1 mM IPTG (isopropyl-ßD-thiogalactopyranoside) and expression of recombinant protein was studied by 15% SDS gel electrophoresis. Recombinant protein was purified by Ni-NTA (Qiagen, USA) affinity chromatography, and concentration was estimated by Lowry's method (Lowry et al., 1951). 2.3. Development of monoclonal antibodies against TRH of V. parahaemolyticus Initially BALB/c mice (6 weeks old) were immunized with 50 μg of TRH recombinant protein in complete Freund's adjuvant (CFA) and three subsequent doses were injected with incomplete Freund's adjuvant (IFA) at 14 day intervals. Antibody titres were determined by plate ELISA. Final booster dose was delivered by injecting the mice intraperitoneally with 100 μL (100 μg) TRH without adjuvant. Hybridoma clones were developed by using the procedure of Kohler and Milstein (1975) with minor modifications. Sensitized spleen cells were fused with mouse myeloma cell line, Sp2/0 using polyethylene glycol after three days from the final booster dose. Hybridoma cells were cloned by limiting dilution and positive clones were selected by plate ELISA. The class of immunoglobulin was determined by using mouse monoclonal antibody Isotyping Reagents (Sigma, USA).

Positive hybridoma clones were cultured in Dulbecco's Modified Eagle's Medium (DMEM) high glucose (Sigma, USA) supplemented with 10% (v/v) fetal bovine serum (Sera Lab Ltd., UK), Antibiotic Antimycotic solution (100×) 1 mL/100 mL (Sigma, USA), Gentamicin (40 mg/mL) 40 μL/100 mL and 2 mM L-glutamine (Sigma, USA). Cells were allowed to grow to form a monolayer in a 37 °C incubator supplemented with 5% CO2. Antibody containing culture supernatants were collected from the hybridoma culture flasks and were centrifuged at 1000 × g for 10 min to remove the cell debris. mAbs were purified using CBindD™ L resin (Sigma, USA) column and the purified antibodies were stored at − 20 °C. 2.6. Monoclonal antibody based sandwich ELISA for the detection of pathogenic V. parahaemolyticus from spiked seafood homogenate All bacterial strains included in this study were used for the evaluation of mAbs. 100 μL of each overnight grown bacterial culture was spiked into 10 g of bacteria free seafood homogenate. Each spiked seafood homogenate was inoculated into 90 mL of alkaline peptone water (APW). Uninoculated sterile seafood homogenate served as a negative control. The enrichment broths were incubated at 37 °C for 16 h and 1.5 mL aliquots of the enriched broth were centrifuged (Heraeus, Germany) at 10,000 ×g for 10 min and the supernatant was used for ELISA. 2.7. Determination of the sensitivity of sandwich ELISA and PCR Two strains of V. parahaemolyticus namely tdh positive O3:K6 obtained from the National Institute of Cholerae and Enteric Diseases (NICED), Kolkata (kindly provided by Dr. T. Ramamurthy) and trh positive AQ4037 obtained from ATCC were used in this study for determining and comparing the sensitivity of mAb based sandwich ELISA and PCR. Finfish (Stolephorus indicus) confirmed to be negative for V. parahaemolyticus was used in this study for preparing the homogenates. 90 mL of APW was added to 10 g of finfish homogenate in a conical flask that was spiked with varying concentrations of pathogenic V. parahaemolyticus to give a final count ranging from 100 to 106 cells/mL. To unspiked finfish homogenate, 90 mL of APW and 1 mL of Luria–Bertani (HiMedia, India) broth was added and served as negative control. All the conical flasks were incubated at 37 °C for 16 h followed by centrifugation at 10,000 × g for 10 min. The resulting supernatants were tested by sandwich ELISA. In parallel, DNA lysate for PCR was prepared by taking aliquots of 1.5 mL of seafood enrichment broths from each flasks and centrifuged at low speed (800 × g for 10 min) (Heraeus, Germany) to sediment the meat particles and the supernatant was centrifuged further at 10,000 × g for 10 min to pellet the bacteria. Pellets were suspended in 100 μL of 1× Tris–EDTA buffer (pH 8.0) and crude DNA was extracted from pellet by heating at 95 °C for 10 min followed by cooling in ice. Aliquots of 2 μL of DNA lysate were used as template DNA for PCR assay. 2.8. Sandwich ELISA for the detection of pathogenic V. parahaemolyticus in seafood homogenate Polystyrene ELISA plates (Greiner Bio-One, Germany) were coated with 100 μL of polyclonal rabbit anti-TRH immunoglobulin (Raghunath

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et al., 2010) developed in our laboratory (1:1000 dilution in 50 mM sodium carbonate–bicarbonate buffer, pH 9.6) and incubated at 4 °C overnight. The unbound active sites in ELISA plates were then blocked with phosphate buffered saline (PBS, pH 7.4), containing 2% bovine serum albumin fractions (HiMedia, India) at 37 °C for 1 h. After rinsing each well with PBS, 100 μL of supernatant from spiked seafood homogenate was added and incubated at 37 °C for 1 h. The plates were washed three times with PBS containing 0.02% Tween 20 (PBS-T) followed by washing three times with PBS. Purified mAb (100 μL) was added to each well and incubated for 1 h at 37 °C. Then plates were washed three times with PBS-T followed by three washings with PBS. All the washing steps were carried out using automated plate washer (Wellwash AC, Thermo Scientific, USA). Each well was incubated with 100 μL of horseradish peroxidase (HRP) conjugated rabbit anti mouse immunoglobulin (Dako, Denmark) in the ratio of 1:2000 in PBS for 1 h at 37 °C. After three successive washings with PBS-T and with PBS, 100 μL of freshly prepared 3,3′,5,5′ tetramethylbenzidine with 30% hydrogen peroxide (TMB/H2O2) (Bangalore Genei, Bangalore, India) was added into each well in the ratio of 1:20. Incubation was carried out for 5 min at room temperature in the dark and the reaction was stopped by adding 100 μL of 5 N H2SO4. Enzyme activity was read at 450 nm in ELISA reader (ELX 800, Bio-Teck Instrument, USA). The absorbance value of each well was compared with positive and negative control included in the assay. The study calculated positive–negative threshold (negative index) using the following formula, negative OD × 2 (2 times the negative optical density). Indices of ≤0.280 were considered as negative. The culture samples were considered positive if its OD was higher than the negative index. 2.9. Detection of pathogenic V. parahaemolyticus from seafood using sandwich ELISA 2.9.1. Sample collection and processing Freshly caught clam, fish, shrimp, and mussel were collected from Mangalore fish landing centre and fish retailers of Mangalore. Total 34 seafood samples were tested for the presence of pathogenic V. parahaemolyticus using mAb based sandwich ELISA and PCR targeting virulence genes tdh and trh. 2.9.2. Sample preparation for ELISA protocol and PCR Ten grams of homogenized meat was added to 90 mL of APW. Flasks were incubated at 37 °C for 16 h and two sets of 1.5 mL seafood enrichment broth aliquots were taken into microcentrifuge tubes separately from each flask and used for preparation of samples for ELISA and PCR assay. Enrichment broths were centrifuged (Heraeus, Germany) at 10,000 ×g for 10 min and supernatant was used for ELISA. Sandwich ELISA protocols were performed using mAb for the detection of V. parahaemolyticus from seafood as described earlier in the text. For preparing samples for PCR, aliquots of 1.5 mL of seafood enrichment broths were centrifuged at low speed (800 × g for 10 min) (Heraeus, Germany) to sediment the meat particles and the supernatant was centrifuged further at 10,000 ×g for 10 min to pellet the bacteria. Pellets were suspended in 100 μL of 1× Tris–EDTA buffer

(pH 8.0) and crude DNA was extracted from pellet by heating at 95 °C for 10 min followed by cooling on ice. Aliquots of 2 μL DNA lysate were used as template DNA for PCR assay. 2.9.3. PCR assay The PCR was performed in a 30 μL mixture consisting of 3 μL of 10× buffer (Bangalore Genei, Bangalore, India), 200 μM each of the four deoxynucleotide triphosphates (dNTPs), 10 pmol of each primer and 1 U of Taq DNA polymerase (Bangalore Genei, Bangalore, India). Two microliters of enrichment lysate was used as DNA template. The reference strain AQ4037 (trh) obtained from ATCC and V. parahaemolyticus (tdh) strain isolated from a case of gastroenteritis at National Institute of Cholerae and Enteric Diseases (NICED), Kolkata, India, were used as positive control and sterile distilled water served as negative control. Thirty cycles of amplification were performed in a programmable thermocycler (MJ Research., USA). The primers used and their respective thermocycling conditions are detailed in Table 2. PCR products were electrophoresed in a 1.5% agarose gel, stained with ethidium bromide (0.5 μg/mL) and photographed using a gel documentation system (HeroLab, Germany). 3. Results 3.1. Cloning and expression of the trh gene V. parahaemolyticus trh gene was amplified and the resultant product was cloned and expressed in SG13009 E. coli cells. The recombinant protein obtained was estimated to have a molecular weight of 25 kDa as determined by 15% SDS-PAGE analysis (Fig. 1A). 3.2. Development of hybridoma clone and characterization A total of six stable hybrid clones were obtained against TRH protein of V. parahaemolyticus. All the clones belonged to IgM isotype (1A2, 4B10, 2C3, 4C3 and 4C12) except for 1B10 which was of IgG isotype. Among the six stable hybridoma clones only 3 clones (1B10, 4B10 and 4C12) detected most of the pathogenic V. parahaemolyticus isolates by plate ELISA. The monoclonal antibodies cross reacted with both TDH (23 kDa) and TRH (25 kDa) recombinant proteins in Western blotting analysis respectively (Fig. 1B). 3.3. Sandwich ELISA for the detection of pathogenic V. parahaemolyticus in seafood homogenate The three mAbs tested could detect all the pathogenic V. parahaemolyticus isolates from spiked seafood homogenate with varying sensitivity. The mean OD values for mAb 1B10 and 4C12 sandwich ELISA were 0.436 and 0.423 respectively, which were lower than the value observed for 4B10 (0.689) (Table 3). All the tested mAb could detect tdh and trh V. parahaemolyticus. Among the three mAbs, 4B10 was observed to show higher sensitivity for the detection of TDH and/or TRH in seafood homogenate enrichments. Hence, it was used in further studies to analyze the seafood samples for pathogenic V. parahaemolyticus.

Table 2 PCR primer pairs and thermocycling conditions for the detection of total and pathogenic V. parahaemolyticus. Primers

Primer sequence

Annealing temperature (°C) and time (min)

No. of cycles

Product size (bp)

References

toxR VP (F) toxR VP (R) tdh D3 (F) tdh D5 (R) trh R2 (F) trh R6 (R)

5′-GTCTTCTGACGCAATCGTTG-3′ 5′-ATACGAGTGGTTGCTGTCATG-3′ 5′-CCACTACCACTCTCATATGC-3′ 5′-GGTACTAAATGGCTGACATC-3′ 5′-GGCTCAAAATGGTTAAGCG-3′ 5′-CATTTCCGCTCTCATATGC-3′

63 °C for 1 min

30

368

Kim et al. (1999)

55 °C for 1 min

30

251

Tada et al. (1992)

55 °C for 1 min

30

250

Note: Initial denaturation at 94 °C for 5 min and final extension at 72 °C for 5 min.

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Fig. 1. SDS-PAGE and Western blot analysis. (A) Lane 1: Purified recombinat TDH (23 kDa), Lane 2: Purified recombinat TRH (25 kDa). (B) Lanes 1 and 2 showing reaction of mAB 4B10 with TDH (23 kDa) and TRH (25 kDa) in Western blot analysis (indicated by arrows) respectively, Lane 3: Induced recombinant SG13009 E. coli, Lane M: PMW-M Protein Marker (Bangalore Genei, Bangalore, India).

3.4. Determination of the sensitivity of sandwich ELISA and PCR Sensitivity of mAb based sandwich ELISA for the detection of pathogenic V. parahaemolyticus from seafood (both tdh and trh) was found to be 103 cells after enrichment for 16 h, where as PCR could detect 101 cells after 16 h enrichment. The sandwich ELISA developed in the study detected only viable V. parahaemolyticus since the secretory protein is the target. 3.5. Detection of pathogenic V. parahaemolyticus from seafood using sandwich ELISA To study the utility of mAb based sandwich ELISA, 34 seafood samples were enriched in APW and tested for the presence of TRH and/or TDH by sandwich ELISA. Among the 34 samples analyzed, 14 (41.18%) were found positive in mAb 4B10 sandwich ELISA. The results of both mAb 4B10 sandwich ELISA and PCR results are summarized in Table 4. Among the 34 seafood samples analyzed, total V. parahaemolyticus were detected in 22 (64.70%) samples, tdh gene was detected in 6 (17.65%) samples and trh gene was detected in 9 (26.47%) samples. All PCR positive samples (either tdh or trh positive) were also positive by ELISA except for one fish sample which was positve for trh by PCR.

ELISA has been employed more frequently for the detection of microorganisms and is regarded as rapid and efficient in detection (Honda et al., 1989; Nybroe et al., 1990; Swaminathan and Feng, 1994; Meer and Park, 1995; Kerr et al., 2001). TDH and TRH of V. parahaemolyticus are extracellular toxins and known virulence factors of pathogenic V. parahaemolyticus (Miyamoto et al., 1969; Honda et al., 1987a,b). Hence, the presence of these hemolysins in enriched cultures is considered as a specific marker for the detection of pathogenic V. parahaemolyticus. Detection of TDH and/or TRH mainly depends on the expression level of tdh and/or trh genes and also favorable culture conditions (Honda et al., 1988; Nishibuchi and Kaper, 1990; Xu et al., 1994). In this study, the mAb developed against recombinant TRH detected all the 30 pathogenic V. parahaemolyticus by sandwich ELISA (Table 1). This sandwich ELISA developed based on the detection of the extracellular toxin TDH and TRH produced by the pathogenic V. parahaemolyticus and therefore appears advantageous since it detects only live cells. In this study the results obtained from the PCR assay couldn't differentiate between live and dead cells. However the limit of detection of pathogenic V. parahaemolyticus was 103 cells in mAb based sandwich ELISA, while in the PCR it was 101 cells after 16 h of enrichment. The use of these two methods shows that, for accurate and sensitive detection, a combination of mAb based sandwich ELISA and PCR would help in not missing the detection of pathogenic V. parahaemolyticus.

4. Discussion Several methods have been developed for the detection of foodborne pathogens including polymerase chain reaction, DNA hybridization technique and immunoassays. Among immunoassays,

Table 3 Mean OD readings of monoclonal antibody sandwich ELISA for the detection of pathogenic V. parahaemolyticus. Monoclonal antibody

Enrichment broth

Mean OD

1B10 4B10 4C12

APW APW APW

0.436 0.689a 0.423

Note: This includes all the pathogenic V. parahaemolyticus isolates tested in this study (21 trh+), (03 tdh+) and (06 tdh+ and trh+). a Significant difference from 1B10 and 4C12.

Table 4 Summary of detection of pathogenic V. parahaemolyticus in seafood by 4B10 monoclonal antibody sandwich ELISA and PCR. Samples analyzed (n)

By mAb based sandwich ELISA

By PCR

Samples positive for pathogenic Vp

Samples negative for pathogenic Vp

toxR+

tdh+

trh+

Clam (15) Fish (13) Shrimp (05) Mussel (01)

06 05 02 01

09 (0.160 ± 0.049) 08 (0.185 ± 0.058) 03 (0.108 ± .0.06) –

09 08 04 01

1 2 2 1

5 4 0 0

(0.789 ± 0.10) (0.598 ± 0.16) (0.540 ± 0.10) (0.570)

“Vp” indicates V. parahaemolyticus. The samples negative for pathogenic V. parahaemolyticus gave absorbance below the threshold value of 0.280. Positive and negative samples were indicated with mean OD ± SD in brackets.

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The study also revealed that the mAb developed against TRH reacted with TDH in Western blotting (Fig. 1B). Cross reaction of monoclonal antibody raised with TDH or TRH has been reported earlier (Honda et al., 1989, 1990). This may be because of the shared antigenic epitopes that are common to TDH and TRH. This clearly demonstrates that the antigenic homogeneity exists among TDH and TRH of V. parahaemolyticus. This assumption is supported by the antigenic peptide analysis of the TDH and TRH based on the method of Kolaskar and Tongaonkar (1990) using antigenic peptide tool (http://imed.med.ucm.es/Tools/ antigenic.pl). The results show that TDH has 9 probable antigenic peptides while TRH has 10. Among these, two antigenic peptides SDEILFVVR and PEYFVNVEA are common to both TDH and TRH. Since the predicted antigenic peptides SDEILFVVR and PEYFVNVEA have almost absolute identity, cross reactivity of mAb raised against TRH of V. parahaemolyticus with TDH can be explained. However, this evidence is not sufficient to determine which antigenic residues among these are recognized by the mAb. Among the three mAbs tested, 4B10 showed higher sensitivity for the detection of TDH and/or TRH of V. parahaemolyticus. By PCR, 6 seafood samples were positive for tdh and 9 were positive for trh gene (Table 4). This supports the earlier observation that, the tropical seafood contains higher prevalence of trh V. parahaemolyticus than the tdh bearing V. parahaemolyticus (Deepanjali et al., 2005; Parvathi et al., 2006; Raghunath et al., 2008). In this study, all samples positive by PCR for tdh or trh were also positive by sandwich ELISA except a sample of fish that was trh positive, this could be due to the presence of non-viable trh V. parahaemolyticus or low expression of trh gene in the strain present in this sample. False positive reactions in PCR due to PCR detection of non-viable V. parahaemolyticus have been reported earlier (Hayashi et al., 2006). Five variants of tdh gene and two variants of trh gene have been reported in V. parahaemolyticus and expression of these genes may vary widely in different strains (Nakaguchi et al., 2004). However, human health risk due to strain with low expression of virulence gene would be minimal and therefore, it can be suggested that ELISA would be useful for the detection of pathogenic V. parahaemolyticus for assessing the risk to seafood consumers. The mAb based ELISA was first developed by Honda et al. (1989) for the detection of pathogenic V. parahaemolyticus from clinical samples. Since their study limited to clinical samples, we endeavored to use ELISA for the detection of pathogenic V. parahaemolyticus from seafood, since the matrix of the food sample is an important issue in these tests. Pathogenic V. parahaemolyticus (tdh and/or trh) is disallowed in seafood samples for export and poses a threat to consumer due to its pathogenic potential. Though the mAb based sandwich ELISA does not differentiate tdh and trh V. parahaemolyticus, the test can have application in routine testing of seafood samples in food labs. In conclusion this study enabled the development of mAb to TRH protein of V. parahaemolyticus and application in an ELISA format for the detection of pathogenic V. parahaemolyticus (tdh and trh positive) in pure culture and seafood enrichments. The results of the ELISA were in close agreement to conventional PCR results. The method has widespread application for detection of pathogenic V. parahaemolyticus in a variety of seafood products with the main advantage being simplicity, high throughput, speed and cost. Realtime PCR is highly sensitive and specific, it would enable the detection of low levels of bacteria after short time enrichment. However, this technique has the shortcoming that it requires specialized equipment and expertise in comparison to relatively simple technique of sandwich ELISA. On the other hand, sandwich ELISA used in this study has the disadvantage of inability to distinguish tdh and trh, which is the single most strength of PCR wherein both the genes can be differentiated. However, the application of simple technique for detection of toxin producing V. parahaemolyticus for seafood risk assessment, sandwich ELISA could find application as a routine test in the hands of a technician.

Acknowledgements The financial support from the Department of Biotechnology Govt. of India towards COE-programme support to the Aquaculture and Marine Biotechnology is gratefully acknowledged.

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